Chronic skin wounds associated with various diseases (e.g., diabetes) and aberrant healing from acute wounding (e.g., hypertrophic scarring) is a major health care burden. Our previous studies show that when transforming growth factor 21 (TGF21) is overexpressed at a level comparable to that found in skin diseases, it induces severe skin inflammation, delayed wound healing and excessive fibrotic response. In contrast, its antagonist, Smad7, has a potent anti-inflammatory effect and accelerated skin wound healing with faster re- epithelialization, reduced inflammation and reduced fibrotic response. Hence, we hypothesize: 1) Smad7 protein delivery to the wound bed can treat impaired skin wound healing. 2) Smad7 directly regulates keratinocyte migration and epidermal re-epithelialization during wound healing through its transcription- and protein-interaction activities. 3) Smad7 regulates secreting molecules in the epidermis involved in granulation tissue formation and stromal remodeling of skin wound healing. To test these hypotheses, Aim 1 will examine if local Smad7 protein delivery promotes healing and remodeling of skin wounds. We will use human Smad7 recombinant protein with a Tat cell permeable tag to treat excisional skin wounds in two impaired wound healing models. First, we will utilize K5.TGF21 transgenic wounds as an inflammation-related impaired healing model, which is an ideal model for validation of the targeted effect of Smad7. Second, we will treat wounds in diabetic mice. Aim 2 will analyze molecular mechanisms by which Smad7 regulates keratinocyte migration. Smad7 transcriptional targets and protein partners regulating epidermal re-epithelialization will be identified and validated under pathological conditions in wound samples generated in Aim 1. Aim 3 will identify Smad7 transcriptional targets affecting the wound stroma. We have generated inducible, epidermal-specific Smad7 mice containing a biotin-tagged Smad7 transgene (Smad7biotin). Excisional wounds from these mice at each specific stage of wound healing will be used for high throughput analyses to identify temporal Smad7 targets affecting the wound stroma. Samples generated in Aim 1 will be used to examine if any of these targets are altered by Smad7 treatment and if in vivo knock down of these targets affects healing. The proposed studies help us further understand pathological mechanisms of impaired skin wound healing, test therapeutic approaches by either local delivery of Smad7 or targeting proteins downstream from Smad7. PUBLIC HEALTH RELEVANCE: Chronic skin wounds affect 6.5 million patients in the United States. This application performs experimental therapeutics to treat impaired skin wound healing and analyzes the molecular mechanisms involved.